Summary The proposed work will advance understanding of enteric disease caused by food-borne, Shiga-toxin- producing enterohemorrhagic E. coli (EHEC) constitute the major US cause of life-threatening hemorrhagic colitis and hemolytic uremic syndrome (HUS). While shiga toxin 2a provides the main virulence factor for intestinal and extra-intestinal disease manifestations, clinical data strongly suggest that Stx-toxemia occurs early in illness and is short-lived. We thus focus on early EHEC-host interactions, especially the complex interactions between EHEC and intestinal mucus. Intestinal mucus represents the first line of defense against enteric pathogens and is the first site for the interaction of EHEC with the human colonic epithelium. Our exciting preliminary data suggest that mucus play an important role in EHEC colonization of the human colon. These data were obtained using a novel model of EHEC infection, termed human colonoids, which are primary colonic epithelial cultures derived from adult stem cells isolated from biopsies of healthy donors. Upon differentiation, human colonoids produce a thick layer of mucus similar to that which is normally present in the human colon. EHEC infection results in the destruction of the mucus layer allowing EHEC to gain access to the colonocyte surface. These data suggest that human colonoids recapitulate EHEC infection and provide unique insights into pathogenesis that differ from other studies performed in human colon cancer cell lines, allowing improved appreciation of the roles of virulence factors and assessment of novel therapeutic targets. We hypothesize that EHEC interaction with colonic mucus is a necessary step in initial colonization of colonic epithelium leading to Stx production and full manifestation of intestinal disease. To test this hypothesis, we propose to use two complementary models of EHEC infection: human colonoids and the rabbits, which is the best animal model that can recapitulate EHEC-induced human intestinal pathologies, including hemorrhagic colitis, to achieve the following Aims: 1. Determine whether mucus degradation by EHEC is necessary to colonize the human epithelium. 2. Determine whether direct interaction between EHEC flagella and mucin(s) provides an initial anchor for EHEC-colonocyte attachment. 3. Determine whether the interaction of EHEC with mucus affects the production and release of the major EHEC virulence factor, Stx2a. The results gained from the proposed experiments will further elucidate the molecular mechanisms for EHEC interactions with human colonic epithelium, particularly the mucus layer, and establish the role of mucus in EHEC pathogenesis.